Many viruses can be produced in large amount only in certain specialized cell types. A classic example is hepatitis C virus (HCV), a single stranded positive RNA virus of the Flaviviridae family (Appel et al., 2006), that can be secreted abundantly only by hepatocytes (Chisari, 2005). The factors responsible for this restriction are largely unknown. In the case of HCV, one clue derives from the demonstration that at least a portion of HCV circulates in plasma in complex with Very Low Density Lipoproteins (VLDL) (Andre et al., 2002; Nielsen et al., 2006), a family of spherical particles that are produced only in liver (Gibbons et al., 2004) to export triglyceride and cholesterol ester into plasma (Gibbons et al., 2000). Although HCV and VLDL circulate together, a role for VLDL in viral assembly or secretion has never been demonstrated.
As for all positive-strand RNA viruses, HCV RNA replication occurs in association with cytoplasmic membranes. In the case of HCV these structures, called ‘membranous webs’, have been visualized in cultured human hepatoma Huh7 cells that harbor a subgenomic replicon of HCV (Gosert et al., 2003; Moradpour et al., 2004). These replicons are engineered HCV RNA molecules that contain essential elements for RNA replication, including the coding sequence for the nonstructural (NS) proteins NS3, NS4A, NS4B, NS5A and NS5B (Lohmann et al., 1999). After transfection into Huh7 cells, the replicon RNA replicates but it does not produce infectious viral particles because it does not encode the structural proteins that are required for assembly and secretion of the virus (Lohmann et al., 1999). The membranous webs that harbor the HCV replication complex have never been isolated and their composition is unknown.
VLDL assembly is currently believed to occur at two different stages (Shelness and Sellers, 2001). In the first stage, Microsomal triglyceride transfer protein (MTP) transfers lipid to nascent apolipoprotein B, a huge 540 Kda protein that gives structural integrity of VLDL (Olofsson and Boren, 2005). Without sufficient lipid binding, apoB becomes ubiquitinated and degraded during translation (Avramoglu and Adeli, 2004). The apoB-containing lipid particles produced in the first stage of VLDL assembly contain only limited amounts of triglyceride (Gusarova et al., 2003). In the second stage, apoB-containing precursor particles are fused with triglyceride droplets in the luminal compartment (Shelness and Sellers, 2001), a step probably facilitated by apolipoprotein E (apoE), another major protein component in VLDL (Mensenkamp et al., 2001). Although not essential for the direct fusion event, MTP is required to transfer triglyceride from the cytosol to the luminal compartment (Shelness and Sellers, 2001). In human and mice, a genetic defect in MTP severely reduces VLDL secretion (Sharp et al., 1993; Raabe et al., 1998). While the first stage of VLDL assembly is known to occur at the endoplasmic reticulum (ER) (Gusarova et al., 2003), the exact location of the second stage remains controversial (Fisher and Ginsberg, 2002).